| Autor: |
Adam J. Hume, Judith Olejnik, Mitchell R. White, Jessie Huang, Jacquelyn Turcinovic, Baylee Heiden, Pushpinder S. Bawa, Christopher J. Williams, Nickolas G. Gorham, Yuriy O. Alekseyev, John H. Connor, Darrell N. Kotton, Elke Mühlberger |
| Jazyk: |
angličtina |
| Rok vydání: |
2024 |
| Předmět: |
|
| Zdroj: |
Pathogens, Vol 13, Iss 1, p 62 (2024) |
| Druh dokumentu: |
article |
| ISSN: |
2076-0817 |
| DOI: |
10.3390/pathogens13010062 |
| Popis: |
Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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